Microbial life protected from frostbite

From Viking (1976) to Phoenix (2008), NASA missions have detected water on Mars, adding enthusiasm to the search for extraterrestrial life.^1,2 Life requires liquid water, but the Phoenix mission revealed that the subsurface water on Mars is frozen, making more urgent the need for antifreeze mechanisms to ensure survival.^3–5 My colleagues and I have started to alter paradigms for the survival of life in frozen environments. Since our discovery that ribonucleic acid (RNA) and teichoic acid molecules (TAs) can function as antifreeze agents,⁶ we realized that our data can contribute to explaining how microbes survive harsh conditions on present-day Earth, such as the polar ice caps, as well as in the Earth's past.

TAs are found in the cell wall of bacteria and the polymer chain extends into the extracellular space. In this manner, they interact with the environment around the cell, providing a first line of defense against cold-induced damage from ice crystals. TAs have been found in the extracellular milieu after release from the cell wall during bacterial division or as a remnant of cell death. Thus, they are thought to be ubiquitous in nature and are found wherever bacteria are present. TAs can be found with lipid tails, so-called lipoteichoic acid (LTA) which is easily purified from bacteria.

‘Snowball Earth’ is one theory that postulates a substantial freezing period over 600 million years ago, where equatorial regions were covered with ice. This event should have been catastrophic for life, but microbial life survived thanks to unknown antifreeze mechanisms. A related theory, the ‘RNA world hypothesis,’ deals with the origins of life itself. Here, the catalytic properties of RNA and the RNA-based ribosome are thought to generate the basic building blocks for life, proteins, and DNA, by chemical reactions of their basic chemical constituents. These reactions are nearly impossible in liquid water because the reactants are highly dispersed throughout the solution.

Antifreeze properties were previously unknown for RNA and TAs. We have demonstrated that RNA and TAs create a highly connected network of small, water-filled cavities between ice crystals. These localize reactants and give them the ability to react with each other through repeated interactions with RNA and ribosome-type assemblies. The microscopic cavities are interconnected by channels that allow the diffusion of reactants/products to the different RNA/ribosome catalytic reaction sites, which increases the reaction efficiency and the variety of products formed.

We verified the presence of liquid water preserved by RNA and LTA by producing fluorescence microscopy images of samples of water at −20°C with and without TA and B. subtitlis bacteria. We prepared samples by placing a drop of solution on a standard glass microscope slide, protected with a cover slip, and placed on a brass manifold. A mixture of water and ethylene glycol was chilled by a refrigerator and pumped through the manifold, allowing us to freeze the microscope slide and maintain the temperature measured with a small wire thermocouple.

First, we dissolved Rhodamine-B dye (Rhod-B) in the water (see Figure 1A). Rhod-B fluoresces brightly in liquid water, but light emission is quenched when frozen, providing a clear distinction between liquid water and ice crystals. The dark regions are occupied by pure water ice crystals. The light magnification in Figure 1A is 100-fold that of the other images (Figure 1B–D), demonstrating that the fluorescence intensity is negligible. When we added LTA to the liquid water and Rhod-B before cooling, channels of liquid water formed, separating the ice crystals, as shown by the strong fluorescence emission of the Rhod-B (see Figure 1B).

Figure 1. Fluorescence microscopy images at −20°C of: (A) Water with Rhodamine-B dye; (B) Water with Rhodamine-B dye and 5% weight/volume (w/v) lipoteichoic acid (LTA); (C) Water with Rhodamine-B dye and B. subtitlis bacteria; and (D) Water with Rhodamine-B dye, 5%w/v LTA and B. subtitlis bacteria.

Compared to Figure 1A, we estimate a nearly 1000-fold increase in light intensity in Figure 1B, calculated from a 10-fold difference in image brightness and the 100-fold intensity enhancement of Figure 1A. This increased intensity was expected, given that we knew from nuclear magnetic resonance and fluorescence spectroscopy that LTA has antifreeze properties. However, the size and shape of the liquid water fraction of Figure 1B was unexpected. One could imagine that large ice crystals would form and segregate the LTA/dye/water into large pockets. Instead, we observe microscopic ice crystals on the scale of a few microns in diameter and length.

Microbial life segregates into water channels. We isolated B. subtilis, a bacterium whose cell wall contains 50% LTA by weight, in stationary phase. We pelleted and resuspended the sample in 10mM NaCl solution to prevent osmotic shock. Rhod-B dye was able to enter the cells and stain the interior. B. subtilis bacteria are 2μm long and enable Rhod-B emission in liquid water. The fluorescence microscope image shows that the bacteria aggregate in clusters and channels between ice crystals (see Figure 1C). From room temperature images (not shown), the bacteria absorb nearly all of the dye from solution. At −20°C, the 10mM NaCl solution is frozen, yet dye emission is observed (see Figure 1C) because of liquid water in the cytoplasm, cell wall (peptidoglycan and LTA), or both. We suspect liquid water in both regions because we know that RNA, found in the cytoplasm, and LTA, in the cell wall, protects liquid water. However, when 5% weight/volume LTA is added to the droplet of B. subtilis, the arrangement changes dramatically so that the bacteria are restricted to areas between ice crystals (see Figure 1D). The bacteria now reside in channels, similar to that seen with LTA (see Figure 1B).

Life requires liquid water. In nature, RNA and TAs can be excreted into the extracellular milieu and would be present from other bacteria that have died and broken apart. The fluorescence microscopy images show that the water is present in long connected channels between ice crystals. These features would enable life to exist at subfreezing temperatures⁷ by alleviating osmotic pressure, allowing the exchange of nutrients/waste products, permitting bacterial motility along the water channels, and permitting the exchange of biochemical signals to allow cell-to-cell communication and quorum sensing. The presence of liquid water would also allow for DNA repair processes and perhaps other enzymatic events.^3,8 Low temperatures would slow normal processes, but some cold-induced enzymes would be able to function.^3,8,9 Even for quiescent microbes, antifreeze LTA and RNA properties may have a survival role for extremophiles found in ice-core samples.^1,2,10–20

In summary, our experiments are important to understand how liquid water could contribute to microbial survival by permitting nutrient/waste exchange and enable the formation of microbial communities along liquid water channels at sub-zero temperatures.⁷ Additionally, we believe that molecular motion of RNA and LTA keeps super-cooled water in a disorder state, preventing its crystallization. This work addresses major questions surrounding the freeze tolerance of microbes after the discovery of viable microbial life deep in the polar ice on Earth. The impact of this work originates from the potential role of RNA and TAs in microbial cryoprotection, filling a void in the molecular biology of bacteria. We are working to determine if extremophilic microbes overexpress RNA or TAs upon freezing. Likewise, additional experiments are planned to study if RNA catalysis can occur in the water pockets. These studies represent the first steps of a long-term project to examine biological life processes in cold environments.